Gluconobacter oxydans is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering G. oxydans, which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from G. oxydans’s genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P3022 and P0943 revealed strong activities in both Escherichia coli and G. oxydans, and the activity of the strongest promoter (P2703) was about threefold that of the other reported strong promoters of G. oxydans. These promoters were used to overexpress SDH in G. oxydans WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P2057 and P2703. This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of G. oxydans for high-value products.The acetic acid bacterium  Gluconobacter oxydans has been widely applied in the industrial production of L-sorbose from D-sorbitol  to overexpress 3-dehydroquinate dehydratase. G. oxydans 621H, and used the constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of inositol dehydrogenase to construct a shuttle vector system. Though some progress were achieved in promoter discovery, it was hard to apply the promoters to other metabolic pathways because the promoters were relatively weak without systematic comparison. Saito et al., found some strong promoters of G. oxydans, such as PtufB, P0169, and P264  in the study of prokaryotic and eukaryotic organisms has become more accessible in the last decade . It has s of PQQ  and the s of PQQ . Kranz eG. oxydans strains that can produce 2-KLG naturally  to overexpress SDH. The results implied the low expression level of SDH may be the main problem for 2-KLG production in many G. oxydans strains. In conclusion, this study obtained a series of gradient promoters, and these promoters revealed promising prospects in metabolic engineering of G. oxydans for high-value products.2-KLG is an important precursor of vitamin C in industry . Howeveraturally , althougis genes . In our dent SDH . Differecofactor . In the Escherichia coli JM109 was used for plasmid construction. G. oxydans WSH-003 was used for PCR amplification of promoters and protein expression. G. oxydans ATCC 621H was used for PCR amplification of promoters. G. oxydans WSH-004 was screened in our previous research  at 30°C with shaking at 220 rpm. Then the cells were harvested and washed twice with PBS. The total RNA was extracted by RNeasy Mini Kit , and ribosomal RNAs were removed by Ribo-ZeroTM rRNA Removal Kits . The RNA sequencing libraries were constructed by TruSeq RNA Sample Preparation Kit v2  and sequenced on MiSeq  using MiSeq Reagent Kit v3 . RNA sequencing was performed by Shanghai Biotechnology Corporation . The abundance of transcripts was determined using bowtie2 . Then different promoters were individually inserted into the plasmid p2-5-mCherry by the one-step cloning kit . The sdh gene and gradient promoters were ligated into the vector pBBR1MCS-5 in the same way. All promoters and genes were verified by Sanger sequencing . All vectors were constructed and amplified in the strain E. coli JM109. The vectors p2-5 and pBBR1MCS-5 were transferred by electroporation into G. oxydans WSH-003 , because it was hard to find a proper RBS with guaranteed strength. Promoters P WSH-003 , which wG. oxydans WSH-003 were picked into 14 mL tubes containing 2 mL of sorbitol medium and cultured for 24 hours at 30°C with shaking at 220 rpm. Then 2% of these cultures were inoculated into a 250 mL flask containing 25 mL of sorbitol medium and cultured at 30°C with shaking at 220 rpm. The cell fluorescence and cell density (OD600) were measured every 4 hours on a Synergy H1 Hybrid Multi-Mode Microplate Reader  with excitation and emission wavelengths of 580 and 610 nm, respectively. The relative activity of mCherry was defined as the ratio of relative fluorescence unit (RFUs) divided by the optical density (OD600). The strain G. oxydans WSH-003 harboring p2-5-mCherry without promoters was used as the control.Single colonies of 3. Single colonies of G. oxydans WSH-003 were picked into 250 mL flasks containing 25 mL of sorbitol medium and cultured for 24 hours at 30°C with shaking at 220 rpm. Then, 10% of these cultures were inoculated into 250 mL flasks containing 25 mL of fermentation medium and cultured at 30°C with shaking at 220 rpm.The fermentation medium was formed with sorbitol medium (50 g/L sorbitol and 10 g/L yeast extract) containing 20 g/L CaCO2SO4 as the eluent at a flow rate of 0.5 mL/min . The concentrations of D-sorbitol, L-sorbose, and 2-KLG were detected by HPLC using an Aminex HPX-87H column  at 35°C with 5 mmol/L H5 mL/min .t tests were performed to demonstrate statistically significant differences between data points. A p-value of ≤ 0.05 was thought to be statistically significant. For data illustration, bar charts with error bars were used.The results were interpreted with mean values and its standard. The paired two-tailed Student G. oxydans WSH-003 were subjected to high-throughput Illumina paired-end sequencing to obtain a global view of the transcriptome after removing ribosomal RNAs. The RNA sequencing data was submitted to sequence read archive (SRA) with the accession number of PRJNA706889. A total of 19.84 million mapped reads with an average length of 100 bp were obtained; 19.31 million reads were uniquely mapped to the genome of G. oxydans WSH-003, which represented a 500-fold coverage of the genome. The transcriptome data were analyzed with the software bowtie2  from the genomes of WSH-003 and ATCC 621H. Compared with these four reported strong promoters, the promoter P2703 has the highest strength, which was about 2.8-fold higher than that of P264 and about 3.1-fold higher than that of Pdnak  in E. coli and G. oxydans were discovered in this study. The E. coli JM109 containing the above plasmids showed visible red fluorescence  value, which may be the reason why these two promoters had high activities in both E. coli and G. oxydans.In this study, a group of promoters with different strengths was obtained based on RNA-Seq data of whole transcripts. The relative strength of these promoters covered a range of about 28 times, from 400 to 11,000, while reported promoters covered about 10 times, from 400 to 4000. Among them, the activity of the newly discovered strongest promoter PE. coli  with different strengths, were generated by random mutation and characterized. In a recent study, a newly tunable L-arabinose-inducible PBAD promoter was discovered to be useful in G. oxydans 621H, and the activity of this promoter was affected by the pH of the medium (G. oxydans for high-value products. With further research, more serviceable promoters of G. oxydans are expected to be discovered and constructed.With the development of synthetic biology, many methods can be applied to improve the strength of promoters, for example, randomization of the non-conserved region of the promoters , error-pe medium . In summhttps://www.ncbi.nlm.nih.gov/biosample/?term=SAMN18147321.The data presented in the study are deposited in online repositories: YC, LL, and SY performed the experiments and data analysis. YC and JZ wrote the manuscript and conceived the study. JL, JZ, and JC coordinated the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that causes COVID-19. Being aware of the presence of the virus on different types of surfaces and in different environments, and having a protocol for its detection, is important to understand the dynamics of the virus and its shedding patterns. In Ecuador, the detection of viral RNA in urban environmental samples has not been a priority. The present study analyzed samples from two densely populated neighborhoods and one public transportation system in Quito, Ecuador. Viral RNA presence was assessed using RT-LAMP. Twenty-eight out of 300 surfaces tested positive for SARS-CoV-2 RNA (9.33%). Frequently touched surfaces, especially in indoor spaces and on public transportation, were most likely to be positive for viral RNA. Positivity rate association for the two neighborhoods and for the surface type was not found. This study found viral RNA presence on urban surfaces; this information provides an insight into viral dissemination dynamics. Monitoring environmental SARS-CoV-2 could support the public health prevention strategies in Quito, Ecuador. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible, positive-sense, single-stranded RNA virus . It is tIn Ecuador, the first official case of COVID-19 was reported in late February 2020, and measures taken by the Ecuadorian government to contain the virus included home confinement with the suspension of all non-essential activities . HoweverThe high transmissibility of SARS-CoV-2 is a key factor explaining the persistence of COVID-19. The primary route of viral transmission is direct person-to-person contact, through respiratory droplets. Additionally, alternative routes of transmission have been proposed via indirect contact through fomites and SARS-CoV-2 positive surfaces, as large amounts of SARS-CoV-2 can cause some virus to remain viable on dry surfaces and in aerosols for long periods of time ,7,8.In previous studies, the stability of SARS-CoV-2 has been assessed in different environmental conditions. In general, under typical indoor environmental conditions, the virus is expected to survive for up to 3 days on non-porous surfaces, such as stainless steel, plastic, and glass . HoweverSARS-CoV-2 RNA detection in the environment could be an indicator of viral shedding . Being aIn Ecuador, SARS-CoV-2 research has mainly been focused on identifying the virus in patient samples and sequencing the genomes of the different variants in circulation ,17,18; hA total of 300 environmental samples were collected in the city of Quito, Ecuador, during August 2020 in two neighborhoods (Belisario Quevedo and Iñaquito) and one public transportation service . The two® LAMP Kit  was used with specific SARS-CoV-2 primers targeting the nucleocapsid (N2) and envelope (E1) genes. Amplification products were visualized in a 1.5% agarose gel  , which inactivates and stabilizes the viral genetic material. An area of 10 × 10 cm was covered with a swab that was then placed in 1.5 mL tubes with 150 uL TRIzol reagent . Samples were stored at −80 °C for downstream processing. The type of surface material and type of environment (indoor/outdoor) for each sample were recorded. RNA isolation was performed using a TRIzol/chloroform-based protocol  . SARS-CoV-2 detection was performed using RT-LAMP according to Rojas et al., 2021. Colorimetric WarmStartrose gel .Chi-square and Fisher’s exact tests were used to analyze the positivity rates between neighborhoods, surface materials, environment types, and sampling sites depending on cell size. When entries included zeros or small numbers, chi-square test was used; otherwise, Fisher’s exact test was employed. Therefore, chi-square test was used for the categories ‘environment type’ and ‘neighborhood’, and Fisher’s exact test for ‘surface material’ and ‘sampling site’. Within the ‘sampling site’ category, chi-square test was used for comparisons between transportation and restaurants/minimarkets, shops/business premises and OPP, while Fisher’s exact test was used for all other comparisons. The strength of association between variables was measured by calculating Cramér’s V coefficient. Parameters were used as categorical independent variables in a logistic regression model to determine the effect of each variable on the presence/absence of SARS-CoV-2 RNA. Separate models were developed for each parameter and the association between variables was expressed as odds ratios (ORs) with the respective 95% confidence intervals (CIs). Bivariate analyses and modeling were performed using R programming software version 3.6.3 .The overall SARS-CoV-2 positivity rate was 9.33% (28/300 samples) . Highly p-value = 0.54), nor was there an association between the positivity rates found on different surface materials . For the first parameter, seven positive samples were found in Iñaquito (7.00%) and four in Belisario Quevedo (4.00%). For the latter parameter, SARS-CoV-2 positive samples were collected from wood , metal , and plastic  surfaces. The samples collected from glass (0/17) and ceramic (0/4) surfaces were all negative. In contrast, we found a significant association between the positivity rate and the type of environment . In this case, the odds of a sample from an indoor environment being positive is 3.36 times higher than the odds of an outdoor environment sample being positive  , where the odds of a sample from the public transport units being positive is 3.31 times higher than the odds of a positive sample being obtained from an OPP  (We did not find a significant association between the positivity rate and the neighborhoods from which the samples were taken ( = 0.01) . The pos = 0.04) .We report a general positivity rate of 9% for our samples A, which In this study, transportation was the sampling site with the highest positivity rate A. SARS-Cp-value = 0.01) between the positivity rate and the type of environment, with a higher probability of obtaining positive samples indoors (3.36 times)  reported that the odds of indoor transmission are very high compared to outdoor transmission (18.7 times). In accordance, we found a significant association 6 times)  28]. Ae. Aep-val times  26 times) B. IndoorWe did not find a significant association between the positivity rates for the two neighborhoods. This result was unexpected, as Belisario Quevedo was one of the neighborhoods with the highest prevalence of COVID-19 cases in Quito at the time this study was conducted; nevertheless, this could be explained by the fact that the entire city was under quarantine and strict vehicular circulation measures . These mThere was no significant association between the positivity rates for different surface material types. Previous studies have suggested that the stability of SARS-CoV-2 varies according to surface type; however, with our results, we can only highlight that SARS-CoV-2 RNA was more commonly found on plastic and metal surfaces. It is important to note that these surfaces comprised most of the analyzed samples ; therefore, this could represent a bias when assessing the association. However, it is important to mention that previous studies assessing viral particle stability on surfaces have found that the virus is more stable on plastic and stainless steel. These studies have found viable particles, under controlled conditions, up to 21 to 28 days following the contact of the virus with the surface, respectively, which could explain our elevated positivity rates on these surfaces ,11.It is important to consider that the present study had some limitations. Firstly, the limit of detection for the technique we employed is 10 viral RNA copies . Therefop < 0.05), especially on public transportation and on highly touched surfaces. Even though indirect viral transmission is now considered to be very low [Our study demonstrates the presence of SARS-CoV-2 RNA on the surfaces in public places and transportation systems located in Quito, Ecuador. We found that there is a higher probability of SARS-CoV-2 RNA being present on indoor surfaces (very low ,33, the very low ,15,34. T


Regarding the validity of squat exercise, the multi-point method (R2 = 0.773) and two variations of the two-point method  showed very large correlations, whereas 40–60% variation showed almost perfect correlation (R2 = 0.962) with the actual 1RM. In conclusion, whereas both the multi-point method and the two-point method load variations showed reliable results, the multiple-point method and most of the two-point methods’ load variations examined in this research provided an accurate (from large-moderate to perfect) estimate of the 1RM. Therefore, we recommend using the multi-point method and especially the two-point methods variations including higher relative loads to estimate 1RM.This study aimed at examining the concurrent validity and reliability of the multi-point method and the two-point method’s variations for estimating the one-repetition maximum (1RM) in the deadlift and squat exercises and to determine the accuracy of which optimal two loads can be used for the two-point method protocol. Thirteen resistance-trained men performed six sessions that consisted of two incremental loading tests  followed by 1RM tests. Both the multi-point method and the two-point method load variations showed reliable results for 1RM estimation (CV < 10%) squat and deadlift exercises. Session-session reliability was found to be low in deadlift (ICC: 0.171–0.335) and squat exercises (ICC: 0.235–0.479) of 40–60% and 60–80% in two-point methods. Deadlift (ICC: 0.815–0.996) and squat (ICC: 0.817–0.988) had high session-to-session reliability in all other methods. Regarding the validity of deadlift exercise, the multipoint method (R SeveralConsidering the limitations of the two methods mentioned above and assuming that the L-V relationship in resistance exercises is stable and linear , the twoe.g., conventional deadlift, squat) has never been explored, and the researchers assume that the inconsistencies in the prediction accuracy of lower body exercises may be due to the technical complexity . For the multiple-point method, each submaximal load and velocity were considered, meanwhile different two-point combinations  were used to predict 1RM.Investigations had been conducted for 6 days separated by 48 h resting periods. On the first day and the fourth day, subjects’ squat 1RM and the deadlift 1RM loads were determined and familiarizations were done for further velocity-based assessments. On the second, third, fifth and sixth day, 1RM tests were conducted while saving barbell velocity data to predict 1RM by using a linear regression equation.Thirteen resistance-trained men participated in the study . Including criteria were performing both exercises with a good pattern, at least 2 years of strength training experience. All subjects were free from any musculoskeletal injuries. Before the study, all subjects were informed of the purpose and risks of the investigation and provided signed informed consent. This study was approved by Yalova University Clinic Researches Ethics Committee  and was conducted in accordance with the Declaration of Helsinki.Athletes performed six testing sessions separated by 48 h each other. Squat assessments were done on the first, the second, and the third day, whereas deadlift assessments were done on the fourth, the fifth, and the sixth day. On the first day, and the fourth day, familiarization sessions were conducted for each exercise. In these sessions, self-determined 1RM’s relative loads were used to determine the actual 1RM score. Subjects began with a standardized warm-up followed by an incremental 1RM test. On the second and the third day for squat, on the fifth and the sixth day for deadlift subjects followed the same warm-up protocol and 1RM assessments. Relative loads that were used in the second, the third, the fifth, and the sixth day were determined from the initial 1RM assessment.Regarding 1RM assessment, before each test, a standardized warm-up consisting of 8 min of jogging, 5 min of lower extremity mobility exercises, and 3 reps of the deadlift and the squat with 20 kg barbell was done. A custom-built power cage  and 20 kg barbell  were used for all 1RM assessments. In the first familiarization session, subjects were taught to perform squat at 90° of the knee joint for the standardization of squat exercise. A string placed between two tripods, set for each individual to make them perform squat at 90° was used for all squat assessments. Each athlete touched the string with their hip in the eccentric phase of squat movement . When thRegarding velocity measurements, barbell velocity data was collected by means of a linear position transducer GymAware , whose validity was reported in previous studies . For eac2) between absolute differences for validity.All the statistical procedures were computed in an Excel spreadsheet designed by p < 0.05 alpha level was used as the significance level.As the level of the relationship, minor (0.0), small (0.1), medium (0.3), large (0.5), very large (0.7), almost perfect (0.9) and perfect (1.0) levels were used . The CV 2 = 0.864) and 2 variations of two-point method  showed very large correlations, whereas other two variations of the two-point method  showed almost perfect correlations with the actual 1RM. Regarding the validity of squat exercise, the multi-point method (R2 = 0.773) and two variations of the two-point method  showed very large correlations, whereas 40-60% variation showed almost perfect correlation (R2 = 0.962) with the actual 1RM.This study aimed at examining the concurrent validity and reliability of the multi-point method and the two-point method for estimating 1RM in the deadlift and squat exercises and to determine the accuracy of which optimal two loads variation can be used for the two-point method protocol. Our main findings revealed that both the multi-point method and the two-point method variations showed reliable results for 1RM estimation (CV < 10%) squat and deadlift exercises. Session-session reliability was found to be low in deadlift (ICC: 0.171–0.335) and squat exercises (ICC: 0.235–0.479) of 40–60% and 60–80% in two-point methods. Deadlift (ICC: 0.815–0.996) and squat (ICC: 0.817–0.988) had high session-to-session reliability in all other methods.2 = 0.864) and two variations of two-point method  showed very large correlation, whereas other two variations of the two-point method  showed almost perfect correlation with the actual 1RM. Regarding the validity of squat exercise, the multi-point method (R2 = 0.773) and two variations of the two-point method  showed very large correlation, whereas 40-60% variation showed almost perfect correlation (R2 = 0.962) with the actual 1RM.Regarding the validity of deadlift exercise, the multipoint method  in deadlift exercise are between acceptable upper and lower limits. However, it shows that the 40–60% and 60–90% variations are not between the acceptable upper and lower limits with real RM percentages. Details of the results are given in Past studies that examined 1RM estimation methods using the L-V relationship reported different results for lower-body  and upper-body (bench press) compound exercises. For example, whereas the general validity results of studies on bench press exercise  report a−1, others have recorded deadlift 1RM velocities as 0.16–0.17 ms−1  accurate estimate of the 1RM. Therefore, we recommend using the multi-point method and especially two-point methods variations including higher relative loads in estimating 1RM. While the two-point method may overcome some of the potential limitations associated with the direct determination of 1RM strength, professionals should also be aware that direct determination of 1RM appears to be the most reliable method in training programs where daily 1RM level is not essential. Lastly, strength and conditioning practitioners and sports scientists should take into account that MV was used for this research and other velocity types like MPV and PV may give different results.10.7717/peerj.13013/supp-1Supplemental Information 1Click here for additional data file.


Various factors related to the sensitivity of non-small cell lung carcinoma (NSCLC) to 5-fluorouracil (5-FU) have been reported, and some of them have been clinically applied. In this single-institutional prospective analysis, the mRNA expression level of five folic acid-associated enzymes was evaluated in surgical specimens of NSCLC. We investigated the correlation between the antitumor effect of 5-FU in NSCLC using an anticancer drug sensitivity test and the gene expression levels of five enzymes.Forty patients who underwent surgery for NSCLC were enrolled, and the antitumor effect was measured using an in vitro anticancer drug sensitivity test (histoculture drug response assay) using freshly resected specimens. In the same sample, the mRNA expression levels of five enzymes involved in the sensitivity to 5-FU were measured in the tumor using real-time PCR. The expression levels and the result of the sensitivity test were compared.DPD), orotate phosphoribosyltransferase (OPRT), or DPD/OPRT expression and the antitumor effects of 5-FU. On the other hand, a correlation was found between thymidylate synthase (TS), folylpoly-c-glutamate synthetase (FPGS), and dihydrofolate reductase (DHFR) expression and 5-FU sensitivity.No correlation was found between dihydropyrimidine dehydrogenase (FPGS and DHFR may be useful for predicting the efficacy of 5-FU-based chemotherapy for NSCLC.Expression of  Therefore, the expression levels of OPRT and DPD may be involved in the effects of 5-FU. In addition, 5,10-CH2-THF, which forms a ternary complex with FdUMP and TS, is one of the reduced folic acids and is regulated by dihydrofolate reductase (DHFR) and folylpoly-c-glutamate synthetase (FPGS) [DHFR and FPGS may also be involved in the effects of 5-FU.5-FU is activated only after it is converted to 5-fluorodeoxyuridine monophosphate (FdUMP). FdUMP inhibits DNA synthesis by forming a ternary complex with Thymidylate synthase (TS), which is an essential enzyme for DNA synthesis, together with 5,10-methylenetetrahydrofolate . It has been reported that the main pathway for conversion of 5-FU to FdUMP in tumors is the pathway converted by Orotate phosphoribosyltransferase (e (FPGS)  ThereforTS, DPD, OPRT, DHFR, and FPGS) of factors associated with 5-FU sensitivity was measured with reverse-transcriptase polymerase chain reaction (RT-PCR), and we used the histoculture drug response assay (HDRA) method to assess sensitivity to 5-FU. We then investigated the correlation between the expression level of each factor and the chemosensitivity result.Several reports of NSCLC cases have described the sensitivity to 5-FU using clinical samples –9, but nTissue samples were collected at the time of surgery from 40 patients who underwent surgery at the Department of Surgery (II), University of Fukui Hospital for NSCLC with a tumor size > 20 mm between January 2012 and December 2015. The experimental use of the chemosensitivity test was approved by the Institutional Research Committee, and the trial was approved by the Institutional Review Board for University of Fukui Hospital. All patients were informed of the nature of this study, and written informed consent was obtained.Fresh specimens were sampled from the primary lesion immediately after surgical resection, immersed in Hank’s solution, and used for in vitro chemosensitivity testing with HDRA. Ten-micrometer-thick slices from the paraffin-embedded specimens were later used for quantitative RT-PCR.2 at 37 °C. 5-FU was provided by Tokyo Chemical Industry Co. Ltd.  and used at 300 μg/ml. After these specimens were histocultured, 100 μl Hank’s balanced salt solution containing 0.1 mg/ml type I collagenase (Sigma) and 100 μl 9.6 mg/ml 3--2,5-diphenyl tetrazolium bromide (MTT) solution dissolved in phosphate-buffered saline were added to each culture well and incubated for another 16 h. Following extraction with dimethysulfoxide, absorbance of the solution in each well was read at 540 nm (control 630 nm) using a microplate reader . Absorbance per gram of cultured tumor tissue (OD/W) was calculated from the mean absorbance of tissue from three culture wells. The tumor tissue weight was determined prior to culture.The HDRA was used as an in vitro drug sensitivity test as previously reported. , 11 CollThe inhibition rate was calculated using the following formula:TS, DPD, OPRT, DHFR, and FPGS) using paraffin-embedded sections of resected NSCLC specimens was performed according to the DTP method of Response Genetics  [The quantitative assay of the five genes of interest  . For eveNY, USA) , 14.TS, DPD, OPRT, DHFR, and FPGS) and an internal reference gene (β-actin) was performed using a fluorescence-based real-time PCR system . The final volume of the reaction mixture was 20 µl. Cycling conditions and the primers and probes were described previously by Matsubara et al. [Quantification of the five genes of interest  values of less than 0.05 were considered statistically significant.Statistical analysis was performed on a personal computer with Stat Mate IV . The correlations between the mRNA expression of genes examined and clinico-pathological parameters were evaluated with the Pearson product moment correlation coefficient. To evaluate the correlation between two variables, linear regression was performed, and the Spearman rank correlation coefficient was calculated. Probability .A test was conducted to examine the results of sensitivity to 5-FU by changing the concentration of CDHP in 29 cases of NSCLC. We found no significant difference in sensitivity between the 5-FU only group  and the group to which CDHP was added . Therefore, this study was performed using only 5-FU. We also examined the toxicity of CDHP by performing a CDHP alone sensitivity test without 5-FU. CDHP alone showed a very low inhibition rate and no antitumor effect .Forty patients (29 men (72.5%) and 11 women (27.5%)) who underwent surgery at our hospital for NSCLC were enrolled (Table The operative procedure was lobectomy in 35 patients (87.5%), segmental resection in two patients (5%), and partial wedge lung resection in three patients (7.5%). The histology included 23 cases of adenocarcinoma (57.5%), 15 cases of squamous cell carcinoma (37.5%), and two cases (5%) of other types of NSCLC. Regarding EGFR mutation and ALK translocation in adenocarcinoma, seven cases were L858R positive, three cases were exon 19 positive, one case was ALK positive, and 12 cases were wild type , 1.200 ± 0.637 (n = 37), 0.706 ± 1.23 (n = 37), 0.885 ± 0.477 (n = 37), and 2.10 ± 2.50 (n = 37), respectively : -0.426, p < 0.01), and those of DPD were weakly correlated with those of OPRT  and FPGS . The mRNA expression levels of FPGS were moderately correlated with those of DHFR .Table DPD, OPRT, or DPD/OPRT and the percent inhibition with 5-FU using the HDRA method   Fig. .Fig. 1NoFPGS and DHFR expression and 5-FU sensitivity results in NSCLC. FPGS showed a stronger correlation with 5-FU sensitivity than DHFR.To the best of our knowledge, this is the first study to show a correlation between FPGS converts intracellular folic acid and folic acid antagonists, such as methotrexate, into polyglutamic acid, which is retained intracellularly for a long time. Polyglutamylation of intracellular 5,10-methylenetetrahydrofolate enables more efficient formation and stabilization of inhibitory ternary complexes involving TS and metabolites of 5-FU, and it may also increase the cytotoxicity of 5-FU.  Two repoDHFR is the target enzyme of methotrexate, which enters the cell, tightly binds to DHFR, and inhibits the reduction of dihydrofolate to tetrahydrofolate.  Only one5-FU is activated only after it is converted to 5-fluorodeoxyuridine monophosphate (FdUMP). TS, an enzyme that is essential for DNA synthesis, methylates deoxyuridine monophosphate (dUMP) and converts it to deoxythymidine monophosphate (dTMP). Therefore, FdUMP is covalently bonded to TS together with 5,10-methylenetetrahydrofolate , which is reduced to folic acid, to form a strong ternary complex, which inhibits DNA synthesis.  FPGS actTS expression and 5-FU sensitivity is the opposite of the results of other reports of NSCLC. [In this study, the positive correlation between f NSCLC. , 20–22 Rf NSCLC. , 24 In Nf NSCLC.  suggestiRegarding the sensitivity of 5-FU, the activities of OPRT and DPD have been well evaluated, and several reports show that they are involved in the sensitivity of NSCLC. , 26 The TS, DPD, and OPRT and 5-FU sensitivity in NSCLC. Eguchi et al. [TS and DPD predict the response to S-1-carboplatin chemotherapy in patients with advanced NSCLC. Nakano et al. [TS and DPD mRNA levels, patients with low DPD tumors receiving 5-FU had significantly better prognosis than those who did not receive adjuvant treatment (p = 0.041). Based on these results, quantification of TS and DPD mRNA levels can predict the efficacy of 5-FU after surgery in patients with NSCLC.Several reports have described the relationship between i et al.  evaluateo et al.  reportedo et al.  investig5-FU is rapidly degraded by DPD. Due to the higher DPD activity in NSCLC compared to other carcinomas,  5-FU aloThere are several reports that The HDRA method correlates well with the susceptibility of NSCLC to anticancer drugs and its clinical efficacy. –30 FurthFPGS and DHFR, whether or not this can reflect in vivo drug effect needs more investigation. Furthermore, studying the relationship between anticancer effects in NSCLC patients who actually received 5-FU and the expression levels of various factors in the tumors is necessary in clinical study. Another limitation is the in vitro use of specimens obtained during surgery. For unresectable advanced NSCLC, small specimens such as those obtained from bronchoscopy should be used. The feasibility of such transbronchial lung biopsy samples is being investigated. Nakajima et al. [TS, DPD, TP, and OPRT mRNA. The feasibility of expression analysis should be evaluated. Clinical application is also expected.The limitations of this study are the small number of cases, the single-institution design, and the in vitro results of the anticancer drug sensitivity test. This study pointed out the correaltionship between in vitro sensitivity of NSCLC samples to 5-FU and mRNA level of a et al.  used a mFew reports have examined the sensitivity of 5-FU in NSCLC. Our study provides results that will be useful for assessing the sensitivity of 5-FU in future clinical applications.FPGS and DHFR may be involved in 5-FU sensitivity.The mRNA levels of five folic acid-associated enzymes involved in the antitumor effect of 5-FU were examined in resected NSCLC tumor specimens with RT-PCR, and the in vitro anticancer sensitivity test was performed. In conclusion, TS, OPRT, and DPD are associated with 5-FU sensitivity. Combined with this result, the folic acid metabolism pathway is also important, and it will be the basis for the development of new anticancer agents using both pathways.Previous studies have reported that


N = 30) 3-week-old Sprague Dawley weanlings were divided equally into five groups; Control, Control EBN, Wi-Fi, Sham Wi-Fi, and Wi-Fi + EBN. 2.45 GHz Wi-Fi exposure and 250 mg/kg EBN supplementation were conducted for 14 weeks. Findings showed that the Wi-Fi group had decreased in spermatogonia mitosis status. However, the mRNA and protein expression of c-Kit-SCF showed no significant decrease. Instead, the reproductive hormone showed a reduction in FSH and LH serum levels. Of these, LH serum level was decreased significantly in the Wi-Fi group. Otherwise, supplementing the Wi-Fi + EBN group with 250 mg/kg EBN resulted in a significant increase in spermatogonia mitotic status. Even though EBN supplementation improved c-Kit-SCF mRNA and protein expression, the effects were insignificant. The improvement of spermatogonia mitosis appeared to be associated with a significant increase in blood FSH levels following EBN supplementation. In conclusion, the long-term Wi-Fi exposure from pre-pubertal to adult age reduces spermatogonia proliferation in the testis. On the other hand, EBN supplementation protects spermatogonia proliferation against Wi-Fi exposure.Children are vulnerable to the radiofrequency radiation (RFR) emitted by Wi-Fi devices. Nevertheless, the severity of the Wi-Fi effect on their reproductive development has been sparsely available. Therefore, this study was conducted to evaluate the Wi-Fi exposure on spermatogonia proliferation in the testis. This study also incorporated an approach to attenuate the effect of Wi-Fi by giving concurrent edible bird’s nest (EBN) supplementation. It was predicted that Wi-Fi exposure reduces spermatogonia proliferation while EBN supplementation protects against it. A total of 30 ( Amid the pandemic COVID-19 outbreak, Wi-Fi has become a daily necessity rather than a privilege . The worIn view of the fact that the testis is a sensitive organ to the RFR emitted by Wi-Fi , the focBefore the onset of the first meiosis during puberty, spermatogonial stem cells (SSCs) located in the seminiferous tubule of the testis should undergo two processes. The first is mitosis for the maintenance of the SSC pool, and the second is differentiation to produce differentiated spermatogonia progenitor . The difThe ligand for c-Kit is known as stem cell factor (SCF), a 45 kDa glycoprotein secreted by Sertoli cells and encoded by Steel (Sl) locus on chromosome 12 in humans and chromosome 10 in mice . InteracBesides c-Kit-SCF interaction, important reproductive hormones also influence spermatogonia proliferation . As the Aerodramus fuciphagus, particularly during the breeding season  supplementation to overcome the expected negative impact of Wi-Fi exposure. EBN is a bird’s nest made of male swiftlet saliva, g season . It is cg season . Our preg season .in vitro study  male Sprague–Dawley rats at the age of 3 weeks with an initial body weight of 45 ± 5 g were acquired from the Laboratory Animal Research Unit (LARU) of the National University of Malaysia (UKM). This 3-week-old male rat pups representing 7-year-old children. Wi-Fi exposure was conducted for 14 weeks until the pups reached 17-week-old. The 17-week-old rats represent 33-year-old humans who have matured reproductive systems. This age comparison was performed according to the pre-pubertal period of rats compared to humans (Thirty (o humans .n = 6) in each group. Each rat was housed individually in a plastic cage (43 cm length × 16 cm wide × 29 cm height) and have the privilege to move freely inside the cage without any movement restriction. The rats were fed with respective food pellets, supplied with clean water ad libitum, and kept in 12 h light: dark cycle at the ambient temperature of 22°C ± 5°C. In this study, all animal procedures were approved by the National University of Malaysia (UKM). The approval reference number for this study was FISIO/PP/2018/SITI FATIMAH/28-MAR./908-MAR.-2018-DEC.-2020.The rats were divided randomly and equally into five groups , with siA Wi-Fi router, namely, TP-LINK AC750 Wireless Dual Band Wi-Fi Router Archer C20 , was used. This Wi-Fi router consists of three omnidirectional antennas. Two antennas generate 2.45 GHz Wi-Fi frequency, while one antenna supports dual frequencies, 2.45 and 5 GHz. Only 2.45 GHz Wi-Fi frequency operating at 802.11b/g/n standards was used.The Wi-Fi router was placed at a 20 cm distance from the animal cages . Wi-Fi gBased on the Maximum Permissible Exposure report of TP-LINK AC750 Wireless Dual Band Wi-Fi Router Archer C20, the specific absorption rate (SAR) is approximately 0.41 W/kg.The raw EBN was obtained from an identified swiftlet’s house at Bera, State of Pahang, Peninsular Malaysia. The collected EBN were then processed, freeze-dried, and provided by Glycofood Sdn Bhd, Malaysia.EBN 250 mg/kg supplementation was given to the Control EBN and Wi-Fi + EBN groups. The dose chosen and the method of supplementation was done based on our previous report .After 14 weeks of Wi-Fi exposure, each animal was euthanized with a Ketamine-Tiletamine-Xylazine (KTX) cocktail intraperitoneally. Blood was drawn immediately from retro-orbital sinus and collected in BD Vacutainer SST II Advance Plus Blood Collection Tube . The blood was left undisturbed for at least 30 min at room temperature, and the collection tubes were centrifuged at 1,500 g for 10 min at 4°C. The obtained serum was aliquoted and stored at −80°C until analysis.Both sides of the testis, epididymis and seminal vesicle were carefully dissected and cleaned from surrounding adipose tissue before weighing. The organ coefficient of each dissected reproductive organ was expressed according to the equation: the wet weight of organ (g)/body weight (g) × 100 . The rigSerial sections of 3-μm thick paraffin-embedded testis were prepared and mounted on poly-L-lysine slides. The sections were deparaffinized in xylene and then rehydrated in graded alcohols.The tissue sections were heated in a microwave oven in pre-heated 10 mM sodium citrate buffer pH 6.0 for 5 min for antigen retrieval and permeabilized with 0.1% Triton-X 100 in PBS for 30 min. The slides were washed three times with PBS before incubation in Blocking One Solution  for 10 min to block non-specific binding. The slides were rewashed with PBS three times. The tissue sections were incubated overnight at 4°C with mouse monoclonal phosphohistone 3 (pHH3) antibody conjugated with Alexa Flour 594  at a dilution of 1:100.After antibody binding, the slides were washed three times with PBS. DNA was counterstained with 10 μg/mL Hoechst 33342 and observed using Nikon Eclipse Ni fluorescent microscope under 400 × magnification. About 10–15 random fields were captured using Nikon Y-T TV. The intensity of the staining of 20 random seminiferous tubules was measured using Image J software v1.52a. The steps were repeated for rectum adenocarcinoma tissue  as a pos®RNA kit . The RNA was eluted with 60 μL of RNase-free water, and its concentration was determined using a NanoDrop DS-11 + Spectrophotometer . Total RNA was converted into a more stable single-strand cDNA using commercially High-Capacity cDNA Reverse Transcription Kits . RNA stabilization was done by mixing 1,000 ng of total RNA in a master mix containing RT buffer, 100 mM dNTP mix, RT random primers, and MultiScribe® Reverse Transcriptase. The reaction was conducted for 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C.Total RNA was extracted from the left testis using the NucleoSpinQuantitative Real-Time PCR (qPCR) reactions were conducted using the PrecisionPLUS qPCR master mix . Dilution of 1:5 and 1:50 cDNA was applied for c-Kit and SCF, respectively. Approximately 1 μL of the respective cDNA dilution and 1 μL of each primer  were addAll reactions were run in triplicate. Each sample’s threshold cycle value (Ct) was measured from the fluorescence signal at the end of every extension cycle. β-Actin was used as a housekeeping gene. The Ct values were used to calculate the relative expression levels compared with the housekeeping gene expression.Standard plots were constructed for each target gene by using specific primers . Each stThe frozen rat testis was thawed and homogenized in radioimmunoprecipitation assay buffer (RIPA) buffer containing protease inhibitors. The samples were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was extracted, aliquoted, and stored at 80°C. Bradford assay was performed to determine protein concentration in each homogenate sample. About 0.2 g of the protein sample was mixed with SDS 5 × loading buffer , incubated at 70°C for 5 min, and centrifuged at 12,000 rpm for 30 s. The proteins were electrophoresed on 10% polyacrylamide gels for c-Kit and 8% polyacrylamide gels for SCF.The membranes were blocked using 5% (w/v) skim milk in Tween-containing Tris-buffered saline  for 1 h. Then it was incubated in rabbit c-Kit polyclonal   and rabbit SCF polyclonal   primary antibodies overnight at 4°C. The membrane was washed with TBST and incubated with goat anti-rabbit Ig-G (H + L) conjugated with horseradish peroxidase (HRP)   for 1 h at room temperature. The membrane was rewashed with TBST, and the immunoreactive bands were detected using the Excellent Chemiluminescent Substrate (ECL) Kit . The membranes were stripped and re-blotted with an anti-β-actin antibody to determine equal loading. Protein detection was then done on the membrane. It was performed using Gel Doc Amersham Imager 600 . Digital images from the latter imager were immediately analyzed using Image J v. 1.52a. The band intensities of the protein were normalized to that of β-actin.Serum testosterone was measured by competitive enzyme-linked immunosorbent assay . Serum FSH and LH were measured by sandwich ELISA . Intra-assay and inter-assay variability (CV) for all ELISA kits was less than 10%.All the assay procedures were conducted in duplicate according to the kit’s instructions. In brief, 50 μL of the standard and serum were pipetted into a 96 well testosterone ELISA plate. The well was immediately added with 50 μL of biotinylated antibody, sealed, and incubated for 45 min at 37°C.For FSH and LH hormone analyses, 100 μL of the standard and serum were pipetted into a 96 well ELISA plate. The plate was sealed and incubated for 90 min at 37°C, and all liquid was removed before adding 100 μL of respective biotinylated antibodies into the well. The well was then incubated for 1 h at 37°C.MyAssays.com.After incubation with the respective biotinylated antibody, all the solution was removed from the well and washed three times. About 100 μL of horseradish peroxidase (HRP)-conjugated avidin was added into each well. The well was further incubated for another 30 min at 37°C and washed five times. The colorimetric reaction was allowed by adding 90 μL of the substrate reagent, covered from light, and incubated for another 15 min. The reaction was terminated by adding 50 μL of the stop solution. Absorbance was recorded at 450 nm. Testosterone, FSH, and LH serum levels were interpolated from an eight-point standard curve plotted according to a four-parameter logistic (4PL) by using post hoc analysis was conducted due to the data’s normal distribution and variance homogeneity. The Kruskal-Wallis H test evaluated immunofluorescence intensity and sperm chromatin integrity because of the lack of normality of the data distribution. A p-value < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 22.0 software . One-way analysis of variance (ANOVA) followed by Tukey’s No significant differences in the organ coefficients of the testis, epididymis, and seminal vesicles were found among the groups .p = 0.000, df = 2)  .p = 0.007) and SCF (p = 0.000) in the Wi-Fi group showed a statistically significant difference compared with those in the Sham group. The c-Kit (p = 0.015) mRNA expression was significantly higher in the Sham group than in the Control group  and SCF (p = 0.996) in the Wi-Fi group showed no significant changes compared with those in the Control group. The c-Kit and SCF protein expression levels increased in the Sham group but were not statistically significant compared with those in Control  and Wi-Fi group . These patterns of protein expression indicate that changes in protein and mRNA levels are somehow parallel.We further examined the protein levels of c-Kit  and SCF p = 0.039) and Sham (p = 0.000) groups compared with that in the Control group  while Sham Wi-Fi group was only exposed to inactive Wi-Fi router (data transfer is off). It seems that the active Wi-Fi router had caused a higher RFR compared to the passive Wi-Fi router. This postulation was consistent with a previous study reported by The greater RFR emitted by an active router may cause the decrease of c-Kit-SCF mRNA expression. On the other hand, the probability of having a lower RFR emitted from an inactive Wi-Fi router may promote a positive effect in the testis and cause the increase of c-Kit-SCF mRNA expression in the Sham Wi-Fi group. The postulation about these findings seems to be consistent with biphasic dose-response explained in radiation hormesis theory . AlthougTo the best of our knowledge, this study is the first to report the association of Wi-Fi exposure with the c-Kit-SCF regulation system in the testis. Although the finding seems to be in line with radiation hormesis theory, we cannot rule out the possibility of the detrimental effect when c-Kit and SCF are overexpressed . We do nSince the protein expression of c-Kit and SCF in both groups was not affected, the communication between c-Kit and SCF has still occurred despite the Wi-Fi exposure given. However, the proliferation of the spermatogonia was significantly reduced in both groups. It thus suggested that the c-Kit-SCF crosstalk may have failed. As there were no changes in both proteins’ expression, the reduction in spermatogonia mitosis may have been related to the protein structural changes upon Wi-Fi exposure. The structural changes of a protein due to EMF exposure have been previously proven by As the regulation of the spermatogonia proliferation involved a very complex interaction with other biomolecules, there is a possibility that factors other than c-Kit-SCF may also have been affected by the Wi-Fi exposure. Given that Wi-Fi exposure was conducted before puberty, major male reproductive hormones may have been affected following the exposure. Findings showed a decrease in gonadotropin, FSH and LH serum levels in Wi-Fi and Sham Wi-Fi groups. Whether serum FSH and LH levels are affected by the distinctive exposure of Wi-Fi, or a sign of a typical negative feedback mechanism remains unclear.However, in terms of FSH action, it is the primary hormone that regulates germ cell numbers by increasing the proliferation of spermatogonia . So, a dThe reduction of both gonadotropin hormones suggested that Wi-Fi exposure has affected the pituitary gland. A similar finding was reported by Kaur & Khera, which reported the decrease of FSH and LH secretion following RFR exposure of a cell phone toward male albino rats . Aside fInterestingly, the serum testosterone level was not affected by Wi-Fi exposure. Despite the low serum LH level, the Leydig cells are still responsive and secrete testosterone. According to Supplementation of 250 mg/kg EBN was given to evaluate its attenuation effect on the Wi-Fi exposure. However, the testis coefficient in the Control EBN group showed a significant decrease compared to the Control group. The same result was recorded in the Wi-Fi + EBN group, which showed a significant reduction in testis and epididymal coefficients. These data indicate that supplementation of EBN 250 mg/kg to the pre-pubertal young has decreased testis and epididymal coefficients. This result contrasts with the supplementation of the EBN 250 mg/kg in the adult rats group, which did not cause any decrease in testis and epididymal coefficients .The decrease in testis and epididymal organ coefficients following EBN supplementation to the young rats was likely due to estradiol (E2) hormone activity. This hormone was previously characterized to present in the EBN extract used in this study . Severalin vitro. EBN capability includes the proliferation of rabbit corneal keratocyte cell culture  axis which caused the reduction in the gonadotropin hormone. This result was accomplished by using a 64 bps ping signal, significantly less than the amount of data traffic we encounter in our daily lives. Email, for example, requires at least 0.5 Mbps, video requires 1.5 Mbps, and HD video or two-way video games demand 4.0 Mbps . A more Attenuation of long-term Wi-Fi exposure in young children with EBN had demonstrated a promising finding where it increases the spermatogonia proliferation despite the Wi-Fi exposure given. However, EBN supplementation at a dose of 250 mg/kg per day on young rats from pre-pubertal age until adulthood was found to have side effects. The side effect includes decreased testis and epididymis coefficient and decreased gonadotropin, LH, and FSH. It is most likely related to the hormone E2, which is also present in the EBN. Therefore, despite the high potential of EBN as a treatment for male infertility, the results presented in this study indicate that there is a need to monitor and verify various health claims made against EBN. This cautionary advice is not only for user’s protection but also for ensuring the EBN can be developed safely for human consumption without any unwanted side effects.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the UKM Animal Ethical Committee (FISIO/PP/2018/SITI FATIMAH/28-MAR./908-MAR.-2018-DEC.-2020).FJ, SI, and KO designed the study. FJ and AZ conducted the data acquisition, interpreted, and analyzed the data. FJ drafted. SI, KO, and CH revised the manuscript and supervised the whole work. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intervertebral disk degeneration (IDD) is caused by nucleus pulposus (NP) degeneration and extracellular matrix (ECM) remodeling and cartilage intermediate layer protein (CILP) expression has been confirmed to be increased in IDD. This study is mainly conducted to clarify the mechanism of CILP in the NP cell degeneration and ECM remodeling in IDD.CILP expression in the degenerated NP tissues and cells is quantified by quantitative real-time PCR and western blot. CILP function is assessed by cell cycle assay, 3--2,5-diphenyltetrazolium bromide assay and flow cytometry, β-galactosidase staining, and the detection of ECM-related molecules aggrecan, collagen type I, collagen type II, MMP-3, and MMP-9 expression is accomplished by qRT-PCR. The potential mechanism is authenticated by dual-luciferase reporter gene assay.CILP was increased in the degenerated NP tissues and cells, and the knockdown of CILP promoted the NP cell cycle, increased cell activity, and repressed cell apoptosis and repressed cell senescence and ECM production. Moreover, miR-330-5p targeted the CILP 3′-untranslated region, and miR-330-5p negatively regulated CILP expression. Moreover, the overexpression of miR-330-5p repressed NP cell degeneration and ECM remodeling to relieve IDD by downregulating CILP.MiR-330-5p represses NP cell degeneration and ECM remodeling to ameliorate IDD by downregulating CILP. Low back pain is a worldwide challenge that poses a serious threat to the physical and mental health of humans and imposes a burden on the social economy , 2. Low Cartilage intermediate layer protein (CILP) encodes cartilage intermediate layer protein and it has been corroborated that the abnormal expression of CILP is associated with a variety of human diseases , 7. ReceOn the basis of the above findings, we in depth probed into the mechanism of CILP in regulating the NP cell degeneration and ECM remodeling in IDD, which might provide new potential biomarkers for the relief of IDD.All the NP tissue samples were from the patients with degenerative disk disease who underwent discectomy after acquiring written informed consent from patients. This study was approved by the Ethics Committee of Liaocheng People’s Hospital.2.NP cells were purchased from Procell  and the cells were put in the Dulbecco’s modified Eagle medium  with the addition of 10% fetal bovine serum  and 1% penicillin-streptomycin at 37 °C, 5% CO−∆∆CT method was conducted to test the relative level of different molecules. All the primers sequences were exhibited in Table Based on the instructions of the reagent manufacturer, TRIzol  reagent was applied to extract the total RNA from the NP tissues and cells. After the quality of RNA were analyzed by a NanoDrop ND1000 UV-VIS spectrophotometer , the high-quality RNAs were transcribed into cDNA using a TaqMan® MicroRNA Reverse Transcription Kit  or a reverse transcription cDNA kit . Subsequently, real-time PCR was conducted on the ABI 7300 Real-Time qPCR system  using an SYBR Green PCR kit . U6 and β-actin were the internal references. The 2The NP tissues and the differently treated NP cells were lysed with RIPA buffer . Next, the proteins were quantified by a BCA Protein Assay Kit , and then the proteins with different molecular weights were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred into polyvinylidene fluoride  membranes. The above membranes were incubated with 5% skim milk for approximately 1 h and further incubated with specific primary antibodies, including anti-CILP  and anti-β-actin  at 4 °C overnight. After being washed about two times, the membranes were further incubated with the secondary antibody  for about 1 h at room temperature. Ultimately, the enhanced chemiluminescence reagents  and Image J  were applied to observe and analyze all the protein bands.6 cells/well) were put in 6-well plates and then cultured for nearly 24 h, and then the above synthetic si-CILP, miR-330-5p mimic, and pcDNA-CILP was transfected into NP cells via Lipofectamine 2000.The si-CILP, miR-330-5p mimic, and pcDNA-CILP were from GenePharma Company . Lipofectamine 2000  was applied to cell transfection. Cell transfection details were as follows: NP cells . Ultimately, FC500 flow cytometry  was applied to assess the cell cycle of NP cells.6 cells. Next, 5 μl of Annexiv-FITC  was added and incubated in darkness at 37 °C for approximately 30 min. Then, propidium iodide (PI)  was added and continue to incubate in darkness for nearly 30 min. FC500 flow cytometry  was applied to test NP cell apoptosis.For the cell apoptosis, the NP cells were washed three times. Seventy percent ethanol was applied to fix these cells and the cell density was adjusted to 1 × 106) with different treatments were put in 6-well plates and cultured. Twenty microliters of MTT solution was put in each well at 37 ° C for approximately 1 h. After that, 150 μl of DMSO was put in each well. The viability of NP cells was quantified at 0, 24, 48, and 72 h through an enzyme-linked immunodetection .MTT assay was applied to assess the viability of NP cells. Specifically, the NP cells (1 × 103) were put in 6-well plates. Then, the medium was discarded and the cells were then fixed in the stain-fixative at room temperature for approximately 15 min, then washed three times with PBS, and incubated in the dyeing working solution at 37 °C and 5% CO2. A microscope  was applied to obtain the images and the Image J software was applied to test the positive cells.Based on the standard procedure of the reagent manufacturer, Senescence Cells Histochemical Staining Kit  was applied to analyze the senescence of NP cells. Specifically, NP cells . Furthermore, the dual-luciferase reporter gene was applied to authenticate this interaction. Briefly, a luciferase reporter vector that contained the potential miR-330-5p binding sites of CILP 3′UTR was constructed, and then the above recombinant vector and miR-330-5p mimic or NC mimic (negative control) were co-transfected into NP cells via Lipofectamine 2000 . After approximately 48 h, the luciferase activity was measured by a dual-luciferase reporter gene assay system .P < 0.05 represented statistical significance.All data were exhibited as mean ± standard deviation. Unpaired Student t test was applied to assess the differences of the two groups, and one-way ANOVA followed by Tukey’s post-test was applied to analyze the differences of more than two groups. Although accumulating evidence authenticates that CILP has a regulatory function in various human diseases , 12, theTo probe into the function of CILP in NP cell degeneration, si-CILP was transfected into the TNF-α-treated NP cells. As exhibited in Fig. Furthermore, the β-galactosidase staining corroborated that the treatment of TNF-α promoted the senescence of NP cells, and the transfection of si-CILP reversed this promotion Fig. A. PrevioNext, we probed into the underlying mechanisms by which CILP played a regulatory function in NP cells. Bioinformatics software (ENCORI) predicted that hsa-miR-330-5p, hsa-miR-378g, and hsa-miR-514a-5p contained the binding sites of the CILP 3′UTR, and we selected hsa-miR-330-5p with the highest score for subsequent experiments, and the binding sites were exhibited in Fig. Furthermore, we probed into how the miR-330-5p/CILP axis affected the development of IDD. MiR-330-5p mimic and/or pcDNA-CILP was transfected into the NP cells. As exhibited in Fig. Furthermore, as exhibited in Fig. IDD causes severe health and socioeconomic burdens and is still short of relieve strategies . Thus, iAccumulating evidence has corroborated that CILP is associated with the occurrence and development of IDD , 17. SimMicroRNAs (miRNAs) affect the expressions of target mRNAs via binding to their target mRNAs . PreviouIn summary, our data authenticated that CILP was elevated in the degenerated NP tissues and cells. Furthermore, the overexpression of miR-330-5p repressed NP cell degeneration and ECM remodeling by downregulating CILP. This study might provide new strategies and directions for the relief of IDD.


Pseudomonas aeruginosa bacteriophages exhibiting different morphotypes: a podovirus (LUZ19) and a myovirus (14-1). We optimized the production process for bacteriophage-loaded powders, with an emphasis on long-term storage under ICH  conditions. D-trehalose-/L-isoleucine-containing bacteriophage mixtures were spray-dried from aqueous solutions using a Büchi Mini Spray-dryer B-290 . A response surface methodology was used for the optimization of the spray-drying process, with the following as-evaluated parameters: Inlet temperature, spray gas flow rate, and the D-trehalose/L-isoleucine ratio. The dried powders were characterized in terms of yield, residual moisture content, and bacteriophage lytic activity. L-isoleucine has demonstrated a positive impact on the activity of LUZ19, but a negative impact on 14-1. We observed a negligible impact of the inlet temperature and a positive correlation of the spray gas flow rate with bacteriophage activity. After optimization, we were able to obtain dry powder preparations of both bacteriophages, which were stable for a minimum of one year under different ICH storage conditions (up to and including 40 °C and 75% relative humidity).In the present study, we evaluated the effect of spray-drying formulations and operating parameters of a laboratory-scale spray-dryer on the characteristics of spray-dried powders containing two  Pseudomonas aeruginosa is a Gram-negative bacterium responsible for a variety of infections, ranging from mild skin infections to life-threatening systemic infections, which are difficult to treat due to the bacterium’s intrinsic and acquired resistance mechanisms to a wide range of antibiotics . Fu. Fup-valout ranged between 29 °C and 40 °C and, as expected, was impacted by Tin . Indeed, Tin is correlated with the temperature at the end of the column (Tout)  . As. Asw/w, cyclone) .w/w and was significantly affected by the L-isoleucine concentration  of LUZ19 after spray-drying , and to limit the titer reduction (log10 pfu/mg). The most important output was bacteriophage titer reduction (log10 pfu/mg). Therefore, we decided to focus on a final dried product characterized by a minimal reduction of the bacteriophage titer (<1 log10 pfu/mg), followed by minimal residual moisture content and, finally, a maximal process yield. For the two bacteriophages, the optimal parameters of the drying process—the inlet temperature and the spray-gas flow—were the same . However, for the L-isoleucine concentration, the optimum concentration for drying LUZ19 was 36.7% w/w, while that for 14-1 was 20.6% w/w . More particularly, we decided to increase the yield of the process (%) as much as possible, to decrease the residual moisture content .Then, the optimized powders were produced and the experimental results were compared with the model predictions for both the 95% prediction interval (PI) low and 95% PI high . The resThe pictures obtained by SEM show at the smallest magnification (×1000), homogeneous particles for the optimized powder particles containing LUZ19 and 14-1 C are folPe) [w/w and 20.6% w/w, respectively).This could be explained by the Peclet number (Pe) . Indeed,Pe) is defined by Equation (1) [D represents the diffusion rate of the dissolved solute and K is the surface evaporation rate:The Peclet number (tion (1) , where DThe evaporation rates of the spray-dried formulations containing D-(+)-Trehalose dihydrate and L-isoleucine were similar, as they were exposed to the same drying conditions. However, D-(+)-Trehalose dihydrate and L-isoleucine have different molecular weights . Thus, it can be assumed that the diffusion rate of L-isoleucine is greater than that of D-(+)-Trehalose dihydrate. Therefore, the Peclet numbers for D-(+)-Trehalose dihydrate and L-isoleucine would also be very different. Due to its high Pe, during drying, L-isoleucine precipitated on the surface of the droplets, forming a hydrophobic layer, which interfered with the diffusion of water and induced the formation of corrugated particles. This sometimes led to the formation of hollow grains  Figure E.We can also observe that the particles are corrugated but particles containgLUZ19 showed a less rough surface than particles containing 14-1 G,H.Particle size and shape may affect many important physical properties, manufacturing processability, and quality attributes of dry powder. A Dx(50) of 2 micrometers for LUZ19 and 14-1 spray-dried particles was observed . Small aw/w of their initial weight. The critical relative humidity was found to be around 40% RH (inflection point), representing the maximum relative humidity that the sample can withstand. Then, a decrease in the mass of the sample was observed. The critical relative humidity was exceeded, resulting in recrystallization of the sample and the expulsion of absorbed water. Subsequently, when the sample was subjected to a reduction in relative humidity (desorption isotherm), the mass of the sample did not change, as it was in crystalline form. Above 60% RH, adsorption became irreversible due to crystallization, and the sample permanently kept its water mass. At +25 °C, there was no significant difference with regard to moisture recovery.DVS was used to evaluate the effect of moisture on the solid-state stability of the powders. For this experiment, only LUZ19 and 14-1 powders produced using optimal spray-drying conditions were evaluated at +5 °C , while our experiment showed a negative influence of L-isoleucine on RMC . From the other side, at +40 °C the activity is stable when the concentration of L-isoleucine is 36.7% but not at 20.6%. L-Isoleucine seems to have a protective role regarding the phage activity. It would be interesting to investigate the activity maintenance over longer time periods and to conduct extensive comparative stability study without and with different concentration of L-isoleucine to better understand its protective role but also with other phages to see if it is phage-specific. Moreover, establishing a scientific base for longer shelf times of dried bacteriophage products at 40 °C will allow for their distribution to developing countries, where the available refrigeration capacity is limited .Podoviridae or Myoviridae P. aeruginosa bacteriophages were produced by spray-drying. A Design of Experiment approach was used, including a response surface, in order to understand the relationships between process/formulation parameters and CQAs. An optimal set of parameters was determined using the obtained models, which was subsequently used in preservative spray-drying runs. For the bacteriophage LUZ19, the retention of activity after drying was proportional to the concentration of L-Isoleucine (with a quadratic effect). However, activity decreased at high L-Isoleucine concentrations when the spray gas flow was low. In contrast, at low L-Isoleucine concentrations, bacteriophage activity after drying decreased with a greater spray gas flow. Unlike LUZ19, for the bacteriophage 14-1, the effect of the spray gas flow, as well as the L-Isoleucine concentration, was much less pronounced. However, we observed a negative effect of L-isoleucine concentration and a positive effect of the spray gas flow on the activity of the bacteriophage 14-1. Finally, the powders remained stable for a minimum 1 year under different ICH conditions. To our knowledge, this is the first study in which bacteriophages have been spray-dried with isoleucine, with excellent biological stability up to 40 °C and 75% RH.Optimized biologically active and stable dry powders containing


Objective: Atrial fibrillation is the most prevalent persistent arrhythmia in patients with hypertrophic obstructive cardiomyopathy. Comparative analyses of the safety and effectiveness of septal myectomy with and without surgical ablation are limited. This study aimed to compare the outcomes of septal myectomy with and without the Cox-maze IV procedure in patients with hypertrophic obstructive cardiomyopathy and atrial fibrillation.Methods: Ninety-four patients with hypertrophic obstructive cardiomyopathy and atrial fibrillation who underwent septal myectomy were analyzed, we divided it into concomitant Cox maze surgery (Cox-maze group) and no concomitant Cox maze operation (no Cox-maze group). Freedom from atrial fibrillation recurrence and all-cause mortality after surgery were assessed.Results: Freedom from all-cause mortality after septal myectomy at 1, 3, and 5 years was 98.5 ± 1.5% each in the Cox-maze group and 90.8 ± 6.3%, 85.1 ± 8.1%, and 85.1 ± 8.1%, respectively, in the no Cox-maze group. Patients in the no Cox-maze group had lower survival, freedom from atrial fibrillation recurrence off antiarrhythmic drugs, and arrhythmia control rate (including patients with successful antiarrhythmic drug conversion) than those in the Cox-maze group . Patients who underwent the Cox-maze IV procedure had lower atrial fibrillation recurrence rate than those who did not . Post-operative increases in left atrial diameters  were associated with atrial fibrillation recurrence.Conclusions: The Cox-maze IV procedure combined with septal myectomy improved mid-term survival and reduced mid-term atrial fibrillation recurrence in patients with hypertrophic obstructive cardiomyopathy and atrial fibrillation. The concomitant Cox-maze IV procedure was associated with a lower atrial fibrillation recurrence in patients with surgical hypertrophic obstructive cardiomyopathy and atrial fibrillation. Hypertrophic obstructive cardiomyopathy (HOCM) caused by left ventricular outflow tract (LVOT) obstruction is the most frequent type of hypertrophic cardiomyopathy (HCM) , and sepFor patients with HOCM complicated with AF, dealing with AF is extremely difficult. Pharmacologic therapy has a limited effect on restoring sinus rhythm (SR) . AlthougDue to concerns about the success rate and safety of the Cox-maze procedure, as well as the cost of the operation, some patients did not undergo the Cox-maze procedure at the same time as septal myectomy, and we retrospectively analyzed the post-operative and follow-up results. Details on the diagnostic criteria of HOCM, AF, and AF recurrence are presented in the n = 54), and a small number of patients underwent left atrial ablation operation (n = 14). This study was approved by the ethics committee of Fuwai Hospital . This study was conducted in compliance with the principles of the Declaration of Helsinki.A total of 94 consecutive patients with HOCM who underwent septal myectomy with and without the Cox-maze procedure (we divided them into the Cox-maze group and no Cox-maze group) at our institution between January 2015 and July 2020 were assessed. For patients in Cox-maze group, most patients underwent Cox-maze IV operation ; subsequently, they were administered amiodarone 200 mg orally, three times a day for 7 days, twice a day for 7 days, and then maintained once a day for 3 months. Thyroid function was monitored regularly during medication administration. Heparin  was administered to prevent thrombosis when pericardial/mediastinal drainage decreased 4–6 h after the operation. Warfarin was administered through the gastric tube at the same time. Heparin was stopped when the international normalized ratio was 2.0–2.5. Warfarin anticoagulant therapy was administered for 6 months and was stopped if there was no AF recurrence.All patients were followed up at the time of discharge, 6 months, 1 year, and yearly thereafter, with physical examination, 12-lead ECG, 24-h Holter monitoring, and echocardiography. We defined the patients whose arrhythmias were controlled as those who maintained sinus rate and those who were successfully converted with drugs after AF recurrence after surgery. The criteria for successful conversion and maintenance of SR were defined as definite SR on 12-lead precordial ECG, and no AF was found in 24-h Holter results. The primary end point of this study was comparison of freedom from atrial arrhythmia recurrence and all-cause mortality between the two groups, atrial arrhythmia recurrence defined as any documented AF, atrial flutter, or atrial tachycardia episode of >30 s after a 3-month blanking period.t-test or the Mann–Whitney U-test depending on whether the data is normally distributed. Categorical data are presented as numbers (percentages) and were compared using the chi-squared test or Fisher's exact test as appropriate. The Kaplan–Meier method was used with log-rank tests to evaluate event-free survival. A Cox proportional hazards regression model was used to adjust for confounding factors. Age, sex, and variables with P < 0.1 in the univariable analysis were included in the multivariate regression model. A two-sided P < 0.05 was considered statistically significant. Statistical analyses were performed using IBM® SPSS® version 22.0 .Normal distribution of continuous variables was assessed by Kolmogorov–Smirnov test. Continuous data are expressed as means ± standard deviations or medians [interquartile ranges (IQRs)] depending on whether the data is normally distributed. These data were compared using Student's P = 0.001) and were more likely to have dyspnoea .A total of 94 consecutive patients were enrolled in the study. There were 54 men (57.45%), 42 patients with persistent AF (44.68%), and 52 patients with paroxysmal AF (55.32%). The median (IQR) age was 53  years, and most patients (72/94) had New York Heart Association (NYHA) functional class ≥III. The baseline clinical characteristics of the study population are shown in P < 0.001) and aortic cross-clamp time  than those in the no Cox-maze group; however, the groups did not differ in terms of mechanical ventilation time, post-operative hospital stay, and incidence of major complications  patients, including left atrial ablation in 14 (14.89%) cases and bilateral ablation in 54 (57.45%) cases, and not combined with the Cox-maze procedure in 26 (27.66%) patients. All patients underwent left atrial appendectomy. Patients in the Cox-maze group had longer cardiopulmonary bypass time  than those in the no Cox-maze group, but this difference was not statistically significant.The pre-operative and post-operative echocardiographic data of patients in the Cox-maze and no Cox-maze groups are detailed in The median (IQR) follow-up was 25 (6–48.5) months. One patient in the Cox-maze group died suddenly 2 months after the surgery. Three patients in the no Cox-maze group died, including one who died due to heart failure 3 months after surgery, one due to liver cancer 2 years after surgery, and one sudden death 1 year after surgery.P = 0.01). There were no significant differences in stroke and thrombotic events (P = 0.073) or other complications between the two groups  , larger post-operative LV end-diastolic diameters , and more likely to have chest pain . Among the 28 patients, 9 successfully recovered to SR after drug control (amiodarone), including 6 in the Cox-maze group and 3 in the no Cox-maze group.There were 28 cases of AF recurrence in all patients , includiP = 0.040 and P = 0.012, respectively).In the Cox-maze group, freedom from AF recurrence off antiarrhythmic drugs (AADs) was 90.7 ± 4.0% at 1 year and 68.7 ± 7.2% at 3 years. The 1- and 3-year arrhythmia control rates (including patients with successful AADs conversion) were 90.3 ± 4.1% and 88.2 ± 4.6%, respectively. In patients in the no Cox-maze group, freedom from AF recurrence off AADs was 59.3 ± 10.0% at 1 year and 50.8 ± 10.2% at 3 years. The arrhythmia control rates were 67.8 ± 9.4% and 67.8 ± 9.4% at 1 and 3 years, respectively. As presented in P = 0.002). Furthermore, post-operatively increased LADs  were associated with AF recurrence (In the multivariate Cox proportional hazards regression analysis, after adjusting for confounding factors, it was found that the AF recurrence rate in patients who underwent the Cox-maze IV procedure was lower than that in patients who did not (hazard ratio, 0.141; 95% confidence interval [CI], 0.042–0.479; currence .Our study showed that the mid-term survival of patients who underwent septal myectomy with the Cox-maze IV procedure was higher than that of patients who did not undergo the Cox-maze IV procedure. With regard to AF recurrence, the SR maintenance of patients who underwent the Cox-maze IV procedure was higher than that of patients who did not undergo the Cox-maze IV procedure.There is a high incidence of AF in HOCM, and the related pathogenesis includes increased left atrial afterload caused by LV diastolic dysfunction, mitral regurgitation caused by SAM sign, atrial myopathy mainly manifested as atrial fibrosis, and atrial ischaemia due to microvascular dysfunction , 15. ManFor patients with HOCM and AF, the curative effect of drug therapy and catheter ablation therapy is not satisfactory , 7, and Our study revealed that freedom from AF recurrence off AADs was 90.7 ± 4.0% at 1 year and 68.7 ± 7.2% at 3 years. Moreover, most patients underwent the biatrial Cox-maze IV procedure, and each ablation line was ablated six times; these results are similar to those of other recent studies. Boll et al.  describePrevious studies , 21 haveThis study has some limitations. This was a single-center retrospective control study, and the sample size was small, as it was limited by retrospective data; thus, it is difficult to perform meaningful statistical analysis. In view of the low incidence rate of HOCM and the small number of such operations, our total surgical volume was relatively high. Previous studies –12 on thIn summary, our study confirmed that the Cox-maze IV procedure combined with septal myectomy improved mid-term survival and reduced mid-term AF recurrence in patients with HOCM and AF. The concomitant Cox-maze IV procedure is associated with a lower AF recurrence in patients with surgical HOCM and AF.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of Fuwai Hospital. The ethics committee waived the requirement of written informed consent for participation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To determine physical and psychosocial well-being of adolescents with type 1 diabetes by self-report and parent report and to explore associations with glycemic control and other clinical and socio-demographic characteristics.Demographic, medical and psychosocial data were gathered from 4 participating outpatient pediatric diabetes clinics in the Netherlands. Ninety-one patients completed the Child Health Questionnaire-CF87 (CHQ-CF87), Centre for Epidemiological Studies scale for Depression (CES-D), and the DFCS . Parents completed the CHQ-PF50, CES-D and the DFCS.1c 8.8% . Compared to healthy controls, patients scored lower on CHQ subscales role functioning-physical and general health. Parents reported less favorable scores on the behavior subscale than adolescents. Fewer diabetes-specific family conflicts were associated with better psychosocial well-being and less depressive symptoms. Living in a one-parent family, being member of an ethnic minority and reporting lower well-being were all associated with higher HbA1c values.Mean age was 14.9 years (± 1.1), mean HbA1c values are related to lower social and family functioning.Overall, adolescents with type 1 diabetes report optimal well-being and parent report is in accordance with these findings. Poor glycemic control is common, with single-parent families and ethnic minorities particularly at risk. High HbA Adolescence is a period of rapid biological changes accompanied by increasing physical, cognitive and emotional maturity that can seriously complicate diabetes regulation. Indeed, adolescents with type 1 diabetes as a group display the worst glycemic control compared to other age-groups , which pDifferent results are found when comparing diabetic adolescents with their healthy peers. Diabetic adolescents tend to report no differences or even better QoL compared to healthy peers -9. Only 1c values [The association of glycemic control with QoL by either adolescent or parent report is inconsistent across studies. Half of the studies investigating the relationship found an association between lower well-being and higher HbAc values -14, whilc values ,12,15-19c values ,11. Morec values ,17.Here we present data from a cross-sectional study on physical and psychosocial well-being of Dutch adolescents with type 1 diabetes and the relationship with clinical parameters, which were gathered at baseline as part of an ongoing randomized controlled trial (RCT) testing the effectiveness of periodic monitoring of Health-related Quality of Life (HRQoL) during outpatient visits. These data allow us to 1) compare reported health status of adolescents with diabetes with healthy peers, 2) examine concordance between adolescent and parent report and 3) explore associations with socio-demographic characteristics and glycemic control. A better understanding of these issues is pivotal to improve quality of care for teenagers with diabetes and optimize clinical outcomes.1c and treatment regimen were recorded form the charts.Participants in the age range 13–17 were recruited from four pediatric diabetes outpatient clinics in the Netherlands, including one academic center (VUmc), by sending an information letter. The study was approved by the Medical Ethical committees of all participating centers and written informed consent was obtained from patients and parents. Those children who did not return the informed consent form were approached by their pediatrician at the next routine appointment and were given the opportunity to participate in the study at that time if they so wished. No time or not interested were the most mentioned reasons for declining participation. All adolescents and their parents received a booklet with questions regarding demographic information and questionnaires to assess physical and psychosocial well-being which they could complete at home and return by mail or in the waiting area at their routine appointment. Height, weight, most recent HbAPhysical and Psychosocial well-being of the adolescents was measured using the 87-item child report version of the Child Health Questionnaire (CHQ-CF87), covering domains of physical, emotional, social and mental health [l health . This quDepression As part of psychosocial well-being we assessed the depressive symptomology of both adolescents and parents with the 20-item Centre for Epidemiological Studies scale for Depression (CES-D) [ (CES-D) , scored The CES-D was initially developed to measure symptoms of depression in adult community studies but has been used in adolescent (diabetes) populations subsequently -24. SimiDiabetes-specific family conflict Each adolescent and parent completed the adapted version by Laffel et al. of the Diabetes-specific Family Conflict Scale to assess the degree of family conflict on 19 management tasks [nt tasks ,27. In tnt tasks . The sco1c < = 7.5%) and sub-optimal glycemic control (HbA1c > 7.5%) were examined using χ2- and Mann-Whitney U-tests. Correlations between HbA1c, the CHQ subscales, CES-D and DFCS were explored with Pearson and Spearman correlation coefficients. Multi-linear regression was used to correlate HbA1c with physical and psychosocial well-being. To correct for demographic and diabetes-related variables, we first entered age, sex, ethnicity, family structure, diabetes duration and hospital, after that forward regression was used for the physical and psychosocial well-being variables and interaction terms. SPSS version 11.0.1 was used to execute all analyses.Substitution of missing values and calculation of the subscale scores was performed according to the manual of the CHQ-CD87 and CHQ-PF50 . We exam1c as between participating and non-participating adolescents, the latter however where more likely to be of another ethnicity and from 1 particular hospital.Of the total 171 eligible subjects, 91 adolescents with type 1 diabetes and their parents consented to participate in the RCT. No differences were found in age, gender or HbA2) than in boys (20.4 kg/m2) (P = 0.04). More boys (29.3 %) than girls used three insulin injections per day (P = 0.04), while more girls (17.1 %) then boys used a pump (P = 0.04). Of all the adolescents participating in the study, 18.7 % lived in a single parent family. Ten participants were of another ethnicity (11 %), half of them of Moroccan descent. The distribution of education levels was comparable to the general Dutch school population.As shown in Table Physical and psychosocial well-being reported by either adolescents or parents did not differ on adolescent's age, ethnicity, diabetes duration, BMI or treatment regimen. Adolescents living in a one-parent family reported more limitations in activities with friends and school work due to behavioral problems  (P = 0.035). Boys only rated their physical functioning (P = 0.02) and bodily pain (P = 0.03) better and their global behavior (P = 0.01) worse as compared to girls. No further differences were found between boys and girls.When comparing the scores of the adolescents in our study to scores from a Dutch school population , we founInterestingly, the scores of the adolescent – parent pairs are largely concordant, as illustrated by the correlations for directly comparable subscales ranging from r = 0.27 for the mental health subscale (P = 0.004) to r = 0.70 for the family activities subscale (P < 0.001). Adolescents rated less behavioral problems (higher scores) than their parents did (P = 0.001), which translated in a significant difference in the psychosocial health summary scale (P = 0.004).As expected, depression scores (CES-D) correlated negatively with the scores for all CHQ-CF87 subscales, ranging from r = -0.20 for change in health to r = -0.67 for the psychosocial summary score  reported scores indicating likely depression (≥16). Adolescents with parents scoring above 16 on the CES-D (indication for depression), did not report more depressive symptoms as compared to adolescents with non-depressive parents. Those 13 parents scoring above the cut-off score of 16, rated the physical (P = 0.005) and psychosocial (P = 0.043) health of their children to be worse compared to parents with no depressive symptoms .As could be expected, more diabetes-specific family conflicts were associated with lower psychosocial well-being and more depressive symptoms Table . Parents1c was 8.8% , with 81% of the adolescents above the recommended 7.5% [1c was not significantly correlated with age, gender, diabetes duration, treatment regimen or BMI. Adolescents with good glycemic control (≤7.5%) reported less family conflicts (P = 0.046) than the others. All adolescents from a one-parent family (18.7%) were among the poorly controlled (> 7.5%), as were all adolescents of another ethnicity (11 %). As shown in Table 1c values were associated with more depressive symptoms and lower psychosocial well-being.Mean HbAded 7.5% . HbA1c w± 1.7; 6.–15.0%, w1c with single parent family , other ethnicity  and the CHQ-CF87 subscale role functioning-behavioral  . In other words: living in a one-parent family or being of another ethnicity is associated with a raise in HbA1c of respectively 1.31 % and 1.43 %. Reporting 10 points less limitations on school work and activities with friends due to behavioral difficulties is associated with a decrease in HbA1c of 0.4%.A linear regression was conducted to explore predictors of glycemic control, showing a significant association of HbAFindings from our study show that adolescents in general function well, i.e. comparable to healthy peers, in concert with previous reports -9. We foIn line with the relatively high CHQ scores, we found the prevalence of depression in our sample not to be elevated. This contrasts with studies reporting two- to threefold higher rates of depression in teenagers with diabetes ,24,32. TIn both parent and adolescent report, lower psychosocial well-being is associated with more depressive symptoms and diabetes-specific family conflicts. This is in accordance with earlier studies which suggest that diabetes-specific family factors are strongly related to quality of life in youth with diabetes ,17. Depr1c levels above 7.5 %. Mean HbA1c levels are in accordance with previous studies in adolescents, the distribution, however, is not further specified in most publications. If a looser criterion of 8.0 % is used, still 66 % is poorly controlled. Persisting high levels of HbA1c throughout puberty will significantly increase their risk of developing complications [1c levels. The strongest association when taking into account demographic and clinical variables was found for those adolescents reporting more limitations in activities with friends and schoolwork due to behavioral difficulties.In more than 80 % of the adolescents in our study, diabetes is suboptimal controlled, defined by HbAications . Our datications ,36. More1c also report inconsistent findings [1c values, as in our study. More and larger longitudinal studies are needed to better understand the complex relationship between psychological functioning and HbA1c in adolescents with type 1 diabetes. Clearly, our study points to the need to address psychosocial issues as integral part of outpatient diabetes care, particularly for poorly controlled adolescents. Psychological, family and educational interventions have shown to be effective in improving well-being and glycemic control in diabetic adolescents and their families, although results are not consistent across all studies [Studies investigating the relationship between physical and psychosocial well-being and glycemic control have reported mixed results -19, withfindings , althougfindings ,24,34,35 studies ,38. The A limitation of our study is the fact that about half of the patients decided not to participate in the RCT, possibly causing positive selection bias. However, poor glycemic control was obvious not a reason for decline. Besides that, the variation among individual adolescents in physical and psychosocial well-being is quite large suggesting that adolescent with good as well as with low well-being participated in our study.In conclusion, Dutch adolescents with type 1 diabetes receiving secondary care overall report a satisfactory quality of life, while diabetes control is suboptimal for the majority of them. The participants seem to have found a balance between an acceptable level of daily diabetes self-management and QoL. The challenge then for health care professionals is to help these young patients and their families to further improve glycemic control without diminishing subjective well-being. Psychosocial risk factors for poor glycemic control were identified, underscoring the importance of a holistic approach to diabetes, particularly in this vulnerable age-group.CES-D Centre for Epidemiological Studies scale for DepressionCHQ-CF87 Child Health Questionnaire-Child Form 87 itemCHQ-PF50 Child Health Questionnaire-Parent From 50 itemsDFCS Diabetes Family Conflict ScaleHRQoL Health-related Quality of LifeQoL Quality of LifeThe author(s) declare that they have no competing interests.MW has coordinated the research, collected and analysed the data and drafted the manuscript, FS, RG and HD have participated in the design of the study, interpreting the results and helped to draft the manuscript, JAB, KH and MH have contributed to the acquisition of the data and the draft of the manuscript. All authors read and approved the final manuscript.